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IntArch Allergy Appl Immunol 1990;92:189-192
Characteristics of MTT as an Indicator of Viability and Respiratory Burst
Activity of Human Neutrophils1
Stephen B. Pruett, Audrey Y. Loftis
Department of Biological Sciences, Mississippi State University, Mississippi State, Miss., USA
Assessment of neutrophil viability and respiratory
burst activity are often important in studies of
the damaging effects of inflammatory neutrophils
(PMN). However, conventional viability assays may
be unreliable due to adherence of activated neutro­
phils to surfaces and due to fragmentation of dead
cells. An in situ assay for viability would solve these
problems. Ideally, respiratory burst activity should
also be measurable with a simple assay using the same
culture conditions. In this report we have investigat­
ed the use of the tetrazolium compound 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium
bromide
(MTT) to assay both parameters. Viable cells reduce
MTT to a formazan which is soluble in aqueous iso­
propanol solution and exhibits an absorbance maxi­
mum at approximately 590 nm [1], This phenomenon
is the basis for a widely used colorimetric cell growth
and viability assay [1-4]. Reduction of tetrazolium
compounds is also used to measure the respiratory
burst of phagocytic cells. The most widely used com­
pound for this purpose is nitroblue tetrazolium
(NBT). This compound is reduced by superoxide
1 Supported by grant AFOSR-89-0361 from the US Air Force
Office of Scientific Research and NIEHS N1H-ES-89-31
which is produced during the respiratory burst [5] and
possibly also by intracellular diaphorases [6], Essen­
tially all the formazan produced from NBT by phago­
cytic cells is intracellular or closely associated with
cells [6].
The chemical similarities between NBT and MTT
and an observation by M. Salin and co-workers [pers.
commun.] that MTT is reduced by PMN prompted us
to investigate the use of MTT to assay both the viabil­
ity and the respiratory burst activity of PMN.
All reagents were obtained from Sigma Chemical
Co. (St. Louis, Mo.), and stock solutions were stored
in small volumes at -80°C until needed. Blood for
isolation of PMN was obtained by venipuncture in­
to tubes with EDTA as anticoagulant. The 5 donors
(2 female and 3 male) were healthy volunteers aged
20-35 years. There were quantitative differences in
MTT reduction by PMN from different donors, but
patterns of survival and response to stimuli were simi­
lar for cells from all donors. PMN were isolated using
a discontinuous Ficoll gradient consisting of 9 ml of
1.077 g/ml Ficoll (Histopaque; Sigma Chemical Co.)
layered over 9 ml of 1.119 g/ml Ficoll in a 50-ml coni­
cal centrifuge tube. Eighteen milliliters of blood was
layered onto the gradient and the tube was centri-
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Abstract. In the present study, we have examined the suitability of 3-[4,5-dimethyIthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) for assessing the viability and respiratory burst activity of human neutrophils.
MTT is a good indicator of the relative viability of neutrophils which have been in culture up to 24 h, if suffi­
cient serum (10% fetal calf serum) is present in the medium and the cells being compared have been in culture
for a similar period of time. However, it is not suitable for exact assessment of percent viable cells in a cell pop­
ulation with low viability. The effectiveness of MTT as an indicator of respiratory burst activity is demon­
strated by the sharp increase in MTT reduction induced by respiratory burst stimuli and by the ability of superioxide dismutase to inhibit 75% of MTT reduction by stimulated neutrophils. Unlike nitroblue tetrazolium
(which is reduced primarily intracellularly) and cytochrome c (which is reduced extracellularly), MTT is ap­
parently reduced both intracellulary and extracellulary by activated neutrophils.
Pruett/Loftis
Cell density, cells/well x 10'5
Fig. 1. Effect of increased serum concentration on the ability of
neutrophils to reduce MTT after 24 h in culture. Neutrophils were
cultured for 2 h (a) or 24 h (b) in medium with 10% FCS before ad­
dition of MTT. Some cultures were stimulated with PM A (200 nM)
immediately after addition of MTT. Absorbance at 610 nm was
measured 2 h after MTT addition. H ,0 2(ImM ) was added to some
wells at the beginning of the culture to assess the effect of decreased
cell viability on MTT production by stimulated neutrophils. The
mean ± SD is shown for duplicate wells of all treatments.
fuged at 600 g for 30 min. PMN isolated from the
lower interface were 96-98% pure as indicated by dif­
ferential counts of stained (Cameo Quick Stain II;
Baxter Scientific) smears. The isolated PMN were
washed twice with calcium and magnesium-free PBS
containing 0.1% bovine serum albumin (low endo­
toxin BSA; Sigma Chemical Co.). All buffers and me­
dia were adjusted to pH 6.9-7.0 immediately before
use. The cells were counted by hemocytometer and
resuspended in assay medium which consisted of
RPMI 1640 with penicillin-streptomycin supplement
and fetal calf serum (FCS) at 1,2, or 10%. The MTT
assay was performed by two different methods. In the
experiment shown in figure 2, the method of Denizot
and Lang [1] was used. Briefly, 10 pi of MTT solution
was added to 100 pi of cell suspension in a 96-well
plate. After incubation, the plate was inverted to re­
move supernates and the formazan crystals which ad­
hered to the plate were resuspended in isopropanol
(200 pi) and absorbance at 610 nm was determined us­
ing a plate reader (Multiskan; Flow Labs). Because of
occasionally disparate results among replicates using
this method, all other experiments employed the
method of Mosmann [2] in which acidified isopropa­
nol is added to the wells without first removirfg the
culture medium. Due to the rapid response to some of
the stimuli used, MTT was added to all wells before
stimuli were added. Preliminary experiments indi­
cated that absorbance at 610 nm reached a maximum
2 h after addition of stimulus. Therefore, absorbance
was measured 2 h after addition of MTT (and stimu­
lus) in all experiments. In some experiments, cell via­
bility was assessed by trypan blue exclusion. If the to­
tal number of cells (live plus dead) at the end of the
culture period was less than the initial cell number,
the difference was assumed to be due to fragmenta­
tion of dead cells, and the ‘missing’ cells were re­
garded as dead when percent viability was calculated
[7].
PMN viability was examined at several time points
in a 24-hour culture by trypan blue exclusion and
MTT reduction. The ability of unstimulated PMN
(2x 105 cells/0.2 ml) cultured in 1 or 2% FCS to re­
duce MTT decreased to nearly undetectable levels by
24 h in culture (results not shown). Trypan blue exclu­
sion indicated that viability also decreased, but 42%
of the cells were still viable at 24 h. Opsonized zymo­
san (OpZy) (0.2 mg/ml) [8] and formyl methionine
leucine phenylalanine (FMLP) (10"6 M) increased
MTT reduction during the first 2 h of culture (absor­
bance at 610 nm for controls = 0.176 ± 0.021; OpZy =
0.368 ± 0.041; FMLP = 0.221 ± 0.015). However, these
agents had little effect on MTT reduction or viability
at 5, 10, or 24 h. In contrast, phorbol myristate acetate
(PMA) caused an initial increase in MTT reduction
followed by a decrease to background levels within
4 h. This paralleled a loss of cellular integrity as ob­
served microscopically, but accurate data could not
be obtained for trypan blue exclusion due to the ad­
herence of treated cells to the wells.
Increasing the serum concentration from 1 or 2%
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190
Reduction of MTTby Human Neutrophils
O PMA
A Control
• PMA/freeze-thaw
▲ Control/freeze-thaw
191
Cell density, cells/well x 10 5
Fig. 2. Comparison of MTT reduction vs. cell density plots for
predominantly live and predominantly dead (freeze-thaw) neutro­
phils. Neutrophils at 2 x 106/ml in medium with 10% FCS were in­
cubated at room temperature for 20 min or placed in a -80°C
freezer for 15 min then thawed in a 37 °C water bath (freeze-thaw)
before being placed in culture wells at the indicated densities. MTT
was added to all wells and PMA (200 nM) to some, and absorbance
at 610 nm was measured 2 h later. Viability of unstimulated cells
was determined by trypan blue exclusion at the same time (2 h after
MTT addition) and is shown above the corresponding plots. Values
shown are means for duplicate wells. Standard deviations were
never greater than 20% of the mean.
Fig. 3. Effect of SOD on generation of extracellular formazan
crystals by neutrophils. Neutrophils (2 x l0 5/well) were treated
with MTT and the cells in a received SOD (625 U/ml) and PMA
(200 nM), whereas cells in b received PMA (200 nAi) but not SOD.
Magnification is 400 x and the micrographs were taken 30 min
after addition of MTT.
partially viable cell population have similar slopes.
The results shown in figure 2 demonstrate that this is
not the case for the predominantly viable and pre­
dominantly nonviable cell populations used in this
experiment. .The slopes are significantly different
(p < 0.05; determined as described by Zar [9]) for these
two populations, suggesting that it would not be valid
to use a standard plot with live cells to determine per­
cent viability of an unknown sample. Thus, MTT can
be a useful indicator of the relative viabilities of PMN
populations cultured under similar conditions, but it
may not be suitable for precise quantitation of per­
cent viability in predominantly nonviable cultures.
Superoxide dismutase (SOD) at 250 U/ml inhib-
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to 10% caused unstimulated PMN to retain most of
their MTT reductive capacity during a 24-hour cul­
ture (fig. 1). However, MTT reduction in response to
PMA decreased by more than 50% as indicated by
comparing results in the 2-hour culture with those in
the 24-hour culture (fig. 1). Cell death induced by ex­
ogenous H2O2 decreased the quantity of MTT reduc­
tion noted in cells stimulated with PMA after 24 h in
culture (fig. 1). The decreases in MTT reduction paral­
leled the diminished numbers of intact cells observed
microscopically and in micrographs (not shown).
The simplest approach in using the MTT assay to
determine the percent viability of an unknown cell
population would be to establish a standard curve of
cell number vs. MTT reduction (absorbance at 610)
using a population of cells with known, high viability.
Given the initial cell number in an unknown sample,
the number of viable cells remaining at a given time
point (and thus, the percent viability) could be deter­
mined by comparison to the standard curve. How­
ever, such a procedure would only be valid if the
standard curve and a similar curve obtained using a
192
References
1 Denizot, F.; Lang, R.: Rapid colorimetric assay for cell growth
and survival. Modifications to the tétrazolium dye procedure
giving improved sensitivity and reliability. J. Immunol. Meth­
ods 89: 271-277(1986).
2 Mosmann, T.: Rapid colorimetric assay for cellular growth and
survival. Application to proliferation and cytotoxicity assays. J.
Immunol. Methods 65: 55-63(1983).
3 Heeg, K.; Reimann, J.; Kabelitz, D.; Hardt, C.; Wagner, H.: A
rapid colorimetric assay for the determination of IL-2-producing helper T cell frequency. J. Immunol. Methods 77: 237-246
(1985).
4 Gerlier, D.; Thomasset, N.: Use of MTT colorimetric assay to
measure cell activation. J. Immunol. Methods 94: 57-63 (1986).
5 Baehner, R.L.; Boxer, L.A.; Davis, J.: The biochemical basis of
nitroblue tétrazolium reduction in normal human and chronic
granulomatous disease polymorphonuclear leukocytes. Blood
48: 309-313(1976).
6 Pick, E.; Charon, J.; Mizel, D.: A rapid densitométrie microas­
say for nitroblue tétrazolium reduction and application of
microassay to macrophages. J. Reticuloendothel. Soc. 30: 581593(1981).
7 Salin, M.L.; McCord, J.: Free radicals and inflammation. Pro­
tection of phagocytosing leukocytes by superoxide dismutase. J.
Clin. Invest. 56: 1319-1323 (1975).
8 Wysong, D.R.; Lyman, C.A.; Diamond, R.D.: Independenpe of
neutrophil respiratory burst oxidant generation from the early
cytosolic calcium response after stimulation with unopsonized
Candida albicans hyphae. Infect. Immun. 57: 1499-1505
(1989).
9 Zar, J.H.: Biostatistical Analysis, pp. 292-295 (Prentice-Hall,
Englewood Cliffs 1984).
Received: March 5, 1990
Accepted after revision: May 21,1990
Correspondence to: Dr. Stephen B. Pruett
P.O. Drawer GY
Mississippi State, MS 39762(USA)
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ited MTT reduction by PMA-stimulated PMN by ap­
proximately 50%, but inhibition was minimal in un­
stimulated cells or with boiled SOD (results not
shown). Higher concentrations of SOD diminished
MTT reduction by unstimulated cells but, once again,
not to as great an extern as noted for PMA-stimulated
cells. The absorbance value for non-activated cells
(3.5 x 105 cells/well) was 0.176± 0.015, whereas the
value in the presence of 1,250 U/ml SOD was
0.106 ±0.005. For PMA-activated cells (1.5 x 105
cells/well), the corresponding values were 0.182
±0.035 and 0.044 ±0.010, respectively. Thus, MTT
reduction by activated PMN is predominantly superoxide-mediated. In addition, the data in figure 1 indi­
cate that at low cell densities, only stimulated PMN
reduce measurable quantities of MTT. Under these
conditions, this assay would exclusively detect MTT
reduced as a result of the respiratory burst. Micro­
graphs taken 30 min after PMA stimulation demon­
strate abundant, extracellular formazan crystals as
well as cell-associated formazan (fig. 3). SOD (625 U /
ml) inhibited formation of most of the extracellular
formazan, but deposits which appeared to be intracel­
lular were still present (fig. 3). This reduction of MTT
both intracellularly and extracellularly contrasts with
the exclusively extracellular reduction of cytochrome
c [6] and the exclusively intracellular reduction of
NBT[6].
In summary, MTT can be used to assess the rela­
tive viability and the respiratory burst activity of
PMN, but may not be suitable for precise quantita­
tion of percent viability of predominantly nonviable
cell populations. It appears to be reduced both intra­
cellularly and extracellularly by activated PMN, and
is, therefore, unique among the agents most com­
monly used to assess the respiratory burst.
Pruett/Loftis
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